Synthetic Biology Platform for Sensing and Integrating Endogenous Transcriptional Inputs in Mammalian Cells

Abstract

SummaryOne of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators.

Engineered systems of inducible anti-repressors for the next generation of biological programming

“…a The NAND operation was achieved with two NOTc operations in PARA. In one channel, I A (9) HQN (from Rondon and Wilson 44 ) regulated GFP via a core O ttg operator; in a second channel, R A (1) KSL regulated GFP via a core O agg operator. In the presence of neither ligand, transcription was fully-permitted in both channels (relative OUTPUT = 1), while in the presence of only one ligand, IPTG or D-ribose, one channel was anti-induced, but, in the other, transcription was fully-permitted (relative OUTPUT~1).…”

“…In principle, this is akin to engineering a computer program to carry out a desired task. Recent advances in synthetic biology have brought the fields of biomolecular, electrical, chemical, and computer engineering far closer [4][5][6] , for instance with the development of biological sensors [7][8][9] , memory [10][11][12] , switches and controllers [13][14][15][16][17] , transistors 18 , counters 19 , oscillators and clocks [20][21][22][23][24] , and equivalents of Boolean logic gates [25][26][27] . In many of these developments, transcriptional regulation is achieved via allosteric transcription factors (TFs) 28 .…”

Multiplexed sgRNA Expression Allows Versatile Single Non-repetitive DNA Labeling and Endogenous Gene Regulation

“…Regarding the capacity of the all-in-one sgRNA expression vector, as the upper limit of the sgRNA number that can be assembled is determined by the carrying capacity of the plasmid, there are several possible ways to enhance the system. Firstly, generating multiple sgRNAs from mRNA (30) or tRNA-flanked sgRNAs (31) can make the construct much shorter because only one promoter is needed for the transcription of multiple sgRNAs. Secondly, since the longest fragment in our all-in-one construct is U6 promoter, using other expressing systems will further increase the capacity of our cloning methods.…”

Synthetic gene circuits and cellular decision-making in human pluripotent stem cells

“…For example, a possible function of a gene circuit would be, if TF1 AND TF2 are expressed then induce TF3, if TF3 AND NOT TF2 AND NOT TF1 is expressed induce TF4 etc. [78]. These functions can become more complex over time as the knowledge of cell state transitions becomes more accurate and the ability to produce larger and more robust gene circuits increases.…”